Inorganic Particle Conjugates

ABSTRACT

The ionic conjugates include an inorganic particle electrostatically associated with a macromolecule which can interact specifically with predetermined chemical species or biological targets.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.09/811,824 filed on Mar. 20, 2001, and claims priority to U.S. PatentApplication Ser. No. 60/190,766 filed on Mar. 20, 2000, each of whichare hereby incorporated by reference in their entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No.DMR-98-08941 from the National Science Foundation and Contract Nos.N0001499WX30470 and N0001400WX20094 from the Office of Naval Research.The government may have certain rights in the invention.

BACKGROUND

This invention relates to ionic conjugates including inorganic particlesand macromolecules, and more particularly to an electrostatic conjugateuseful in detecting the presence or absence of specific species, such asfor detecting a biological target.

Labeling of biological molecules using fluorescent tags is a common anduseful practice in biological science. Fluorescent small molecules(conventional organic dyes) are used in both single and simultaneousmultiple detection approaches. However, biological tagging using organicfluorophores has significant limitations. Fluorescent molecules tend tohave narrow absorption spectra and their emission spectra are usuallybroad and exhibit red tailing, making simultaneous quantitativeevaluation of relative amounts of different probes present in the samesample difficult due to spectral cross talk between various detectionchannels. Furthermore, any desired variations of the absorption and/oremission spectra of tagged bioconjugates require the use of distinctmolecular labels with attendant synthesis and bioconjugation challenges.Nonetheless, the use of multiple labels has achieved a considerablelevel of sophistication, as demonstrated by recent flow cytometry workinvolving a three-laser system and eight-color marking scheme tosimultaneously measure a total of 10 parameters on cellular antigens.

SUMMARY

An ionic conjugate forms through self-assembly in which inorganicparticles electrostatically attach associate with at least onemacromolecule. One type of association is self-assembly. Self-assemblyis a coordinated action of independent entities under distributed (i.e.,non-central) control to produce a larger structure or to achieve adesired group effect. Instances of self-assembly occur in biology, e.g.,embryology and morphogenesis, and in chemistry, e.g., the formation ofmore loosely bound supramolecular structures from groups of molecules.Self-assembly of ionic conjugates is driven by noncovalent binding suchas electrostatic interactions between charged, ionizable, or chargeablelinking groups of the inorganic particles and complementary groups ofthe macromolecule.

Each of the macromolecules can also include a moiety that reacts with orexhibits an affinity for a predetermined chemical species or biologicaltarget. For example, the macromolecule can include an antibody,polynucleotide, or cell membrane having a charged, ionizable, orchargeable linking group. Alternatively, a macromolecule that does notreact with or exhibit an affinity for a predetermined chemical speciesor biological target can be attached to a biological moiety with suchproperties. In this instance, the macromolecule portionelectrostatically self-assembles with the inorganic particle and thebiological moiety interacts with a predetermined chemical species orbiological target. As a result, the macromolecules forming theself-assembled supramolecular structures can be preselected so that theionic conjugate will include a macromolecule that will directly orindirectly react with or exhibit affinity for a specific species.

Inorganic particles such as semiconductor nanocrystals provide asolution to many of the problems encountered by organic small moleculesin fluorescent tagging applications, by offering advantages such as ahigh photo-bleaching threshold, excellent chemical stability, andreadily tunable spectral properties. Combining the size-dependentluminescence emission properties of the nanocrystals, their wide rangeof useful excitation and emission wavelengths, resistance tophoto-bleaching, and a high quantum yield in aqueous solutions (highsensitivity) makes these materials very attractive for the labeling ofbiological targets via a self-assembled nanocrystal-macromolecule inwhich the macromolecule contains a moiety having an affinity for aspecific biological target.

In one aspect, the invention features a composition including aninorganic particle, a linking group which has a distal end and aproximal end, the distal end being bound to an outer surface of theinorganic particle and the proximal end including a first charged orionizable moiety, and a macromolecule having a second charged orionizable moiety, in which the first and second charged or ionizablemoieties associates the inorganic particle electrostatically with themacromolecule to form an ionic conjugate. The macromolecule can be afusion protein having a second charged or ionizable moiety, wherein thefirst and second charged or ionizable moieties electrostaticallyassociates the inorganic particle with the fusion protein to form anionic conjugate.

In another aspect, the invention features a method of forming an ionicconjugate by providing an inorganic particle including a linking grouphaving a distal end and a proximal end, the distal end being bound to anouter surface of the inorganic particle and the proximal end including afirst charged or ionizable moiety; and contacting a macromolecule havinga second charged or ionizable moiety with the inorganic particle, inwhich the first and second charged or ionizable moietieselectrostatically associate the inorganic particle with themacromolecule to form an ionic conjugate. The method can further includecontacting a plurality of macromolecules, each of the macromoleculesincluding a charged or ionizable moiety, with the inorganic particle toelectrostatically associate the plurality of macromolecules with theinorganic particle via a plurality of inorganic particle linking groups.The macromolecule can be formed by recombinant or synthetic methods, orisolated from a natural source. Each member of the plurality ofmacromolecules can be the same or different species.

In another aspect, the invention features, a method of detecting thepresence of a predetermined species in a solution. The method includescontacting a solution with an ionic conjugate, in which the ionicconjugate includes an inorganic particle electrostatically associatedwith a macromolecule, the macromolecule capable of binding specificallyto the predetermined species. The method can further includes forming anionic conjugate by adding an inorganic particle and a macromolecule tothe solution. The inorganic particle includes a linking group having adistal end and a proximal end, the distal end being bound to an outersurface of the inorganic particle and the proximal end including a firstcharged or ionizable moiety. The macromolecule includes a second chargedor ionizable moiety. The first and second charged or ionizable moietiesassociate electrostatically to form the ionic conjugate.

Embodiments of the invention may include one or more of the following.The inorganic particle can be a semiconducting nanocrystal (QD). Thesemiconductor nanocrystal can include a first semiconductor materialselected from the group consisting of a Group II-VI compound, a GroupII-V compound, a Group III-VI compound, a Group III-V compound, a GroupIV-VI compound, a Group compound, a Group II-IV-VI compound, and a GroupII-IV-V compound. The first semiconductor material can be selected fromthe group consisting of ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe,HgTe, AlN, AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, GaSe, InN, InP, InAs,InSb, TlN, TlP, TlAs, TlSb, PbS, PbSe, PbTe, and mixtures thereof. Thefirst semiconductor material can be CdSe. The first semiconductormaterial can be overcoated with a second semiconductor material. Thesecond semiconductor material can be ZnS, ZnO, ZnSe, ZnTe, CdS, CdO,CdSe, CdTe, MgS, MgSe, HgO, HgS, HgSe, HgTe, AlN, AlP, AlAs, AlSb, GaN,GaP, GaAs, GaSb, GaSe, InN, InP, InAs, InSb, TIN, TlP, TlAs, TlSb, PbS,PbSe, PbTe, SiO₂, or mixtures thereof.

The inorganic particle can include a plurality of linking groups eachindependently including a charged or ionizable moiety. The ionicconjugate can include a plurality of macromolecules, each of themacromolecules including a charged or ionizable moiety. The plurality ofmacromolecules can associate with the inorganic particle viaelectrostatic interaction with the plurality of inorganic particlelinking groups. The inorganic particle can include Ag, Au, or aphosphor. The first and second charged or ionizable groups can includehydroxide, alkoxide, carboxylate, sulfonate, phosphate, phosphonate, orquaternary ammonium. The second charged or ionizable moiety can be aleucine zipper. The second charged or ionizable moiety can bepolyaspartate. The macromolecule can include a polypeptide orpolynucleotide, such as a maltose binding protein or an immunoglobulin Gbinding protein.

The linking group can have the formula:

(R₁)_(a)—R₂—[(R₃)_(b)(R₄)_(c)]_(d)

wherein

R₁ is selected from the group consisting of C1-C100 heteroalkyl, C2-C100heteroalkenyl, heteroalkynyl, —OR, —SH, —NHR, —NR′R″, —N(O)HR,—N(O)R′R″, —PHR, —PR′R″, —P(NR′R″)NR′R″, —P(O)R′R″, —P(O)(NR′R″)NR′R″,—P(O)(OR′)OR″, —P(O)OR, —P(O)NR′R″, —P(S)(OR′)OR″, and —P(S)OR, whereinR, R′, R″ are independently selected from the group consisting of H, abranched or unbranched C1-C100 alkyl, a branched or unbranched C2-C100alkenyl, a branched or unbranched C2-C100 alkynyl, a branched orunbranched C1-C100 heteroalkyl, a branched or unbranched C2-C100heteroalkenyl, a branched or unbranched C2-C100 heteroalkynyl, with theproviso that when a is greater than 1 the R₁ groups can be attached tothe R₂ or R₃ groups at the same or different atoms within those groups,the R₁ groups can be the same or different, or the R₁ groups can form asix, seven, eight, nine, or ten membered cycloalkyl, cycloalkenyl,heterocyclic, aryl, heteroaryl, or a six- to thirty-membered crown etheror heterocrown ether;

R₂ is selected from a bond, a branched or unbranched C2-C100 alkylene, abranched or unbranched C2-C100 alkenylene, a branched or unbranchedC2-C100 heteroalkenylene, cycloalkyl, cycloalkenyl, cycloalkynyl,heterocyclic, aryl, and heteroaryl;

R₃ is selected from a branched or unbranched C2-C100 alkylene, abranched or unbranched C2-C100 alkenylene, a branched or unbranchedC2-C100 heteroalkenylene, cycloalkyl, cycloalkenyl, cycloalkynyl,heterocyclic, aryl, and heteroaryl;

R₄ is selected from the group consisting of hydrogen, a carboxylate, athiocarboxylate, an amide, a hydrazine, a sulfonate, a sulfoxide, asulfone, a sulfite, a phosphate, a phosphonate, a phosphonium ion, analcohol, a thiol, an amine, an ammonium, an alkyl ammonium, a nitrate;and

a is 1 to 40, b is 0 to 3, c is 1 to 30, d is 1 to 3, and when d is 2 or3 the R₃ groups can be the same or different or can be linked togetherto form a five to ten members cycloalkyl, cycloalkenyl, heterocyclic,aryl, or heteroaryl. The linking group can have the formulaHS—C₂H₄—CH(SH)—(C₄H₈)—COOH.

In another aspect, the invention features a method of forming an ionicconjugate from a modified inorganic particle and a macromolecule. Boththe particle and molecule include charged or ionizable linking groupswhich together can form complimentary ionic pairs to electrostaticallyattach at least one macromolecule to the particle. The inorganicparticle can be modified by bonding one charged or ionizable linkinggroup to the particle surface. The macromolecule can be a modifiedprotein, such as by a recombinant protein process, to incorporate oneend of a charged or ionizable linking group, such as a chargeablepolypeptide, onto the protein's surface.

In another aspect, the invention features, a recombinant proteinelectrostatically attached to a semiconducting nanoparticle. Therecombinant protein can be a fusion protein including any proteinimparting biological activity which has been modified, for example, toinclude a basic leucine zipper. The leucine zipper is a chargeablepolypeptide having one end bound to the protein and another end unboundand protruding away from the surface of protein. The zipper alsoincludes a thiol group which can form a covalent bond with the thiolgroup of a leucine zipper of another fusion protein to form a dimer offusion proteins. The semiconducting nanoparticle can includedihydrolipoic acid groups which electrostatically interact with theunbound end of the basic leucine zipper to form the ionic conjugate.

In another aspect, the invention features a nanoparticle having a coreand overcoat. During routine preparation, an inorganic core of thenanocrystal is capped with an organic shell such as a trioctyl phosphineand trioctyl phosphine oxide mixture (TOP/TOPO), which can be furthermodified and thereby, permit post synthesis manipulation and tailoringof particle solubility in various solvents. Overcoating the CdSe core,for example, with a larger band gap semiconducting material, e.g., ZnSor CdS, a process based on the concepts of band-gap engineering used inelectronics, permits passivation of core surface states and reduces theleakage of excitons outside the core. This overcoating enhances thephotochemical stability of these materials and improves the luminescencequantum yield substantially without affecting the wavelength and thespectral width of the emission, i.e., CdSe—ZnS nanoparticles have anFWHM ˜40-60 nm. In addition, overcoating can enhance resistance tophoto-bleaching. The above properties enhance the sensitivity ofdetection approaches employing these nanoparticles for signalgeneration. Substantial PL intensities with good signal-to-noise ratios,along with well-resolved spectra, are measured for dispersions withconcentrations much smaller than 1 nanomole of nanoparticles per liter.Replacing the TOP/TOPO cap with polar terminated groups allows dispersalof these core-overcoat nanoparticles in aqueous solutions withpreservation of a high photoluminescence quantum yield. Presence of adihydrolipoic acid provided stable water dispersions of CdSe—ZnSnanoparticles with quantum yields of 15-20%. The dihydrolipoic acidgroups on the surface of the nanoparticles are charged or ionizablegroups which electrostatically attach to complimentary charged orionizable groups of a macromolecule.

The ionic conjugates of this invention exhibit biological activity, arechemically stabile, and exhibit increased quantum yield relative toinorganic particles lacking an electrostatically bound protein. Theconjugates also unexpectedly exhibit reduced aggregation relative tobiological conjugates in which a biological moiety is covalently bounddirectly to the inorganic particle. Another advantage of the process ofproducing ionic conjugates of this invention is its simplicity andversatility. For example, the desired protein attaches to the surface ofthe inorganic particle nearly instantaneously.

Ionic conjugates including CdSe nanocrystals overcoated with ZnS anddithiol capping groups and self-assembled with macromolecules offerseveral advantages: 1) because each dithiol-capping molecule can attachto two surface atoms, a higher surface passivation can be achieved withequal or even smaller density of capping groups per unit area, incomparison with mono-thiol capping molecules, for instance. 2) Thecarboxylic acid groups, which permit dispersion of the nanocrystals inwater solutions at basic pH, also provides a surface charge distributionthat can be used to directly self-assemble (or react) with othermacromolecules having a net positive charge. 3) The ZnS coverageprovides a better shielding of the CdSe core from the polar environmentand a more efficient confinement of the exciton (electron-hole pair),which results in stable and highly luminescent nanocrystal dispersionsin water. Dispersions of core-overcoat nanocrystals in water that arestable over a long period of time (several months) and aphotoluminescence quantum yield of ˜20% are easily prepared using theabove approach. 4) The synthetic approach can be easily applied to anumber of different core-overcoat nanocrystals and extended to othercombinations of semiconducting materials, II-VI and III-V, which cangenerate a group of fluorescent probes that can be spectrally tuned.This contrasts with the need of developing specific chemistry routes foreach organic fluorescent dye case-by-case. 5) The synthesis of amacromolecule such as fusion proteins based on the construction vectorstrategy provides a general and consistent scheme to prepare a wideselection of biological macromolecules that can perform specificfunctions and self-assemble with the nanocrystals. 6) The recombinantprotein approach permits one to perform alterations of charged orionizable portion of the macromolecule, e.g., charge, size, stability topH and temperature, and thereby allow one to vary and control theself-assembly of the macromolecule such as to form monomers, dimers andtetramers of macromolecules which can self-assemble onto the inorganicparticle. Each of the macromolecules can include moieties havingaffinities for the same or different biological agents. 7) Controllingthe properties of the peptide tail permits these proteins to interactnon-covalently with a variety of materials (e.g., inorganic colloidalparticles and even surfaces) that have opposite charge to those on thelinker tail of the proteins.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic view of an ionic conjugate of this invention.

FIGS. 2A-2B are schematic cross-sections of inorganic particles of theionic conjugates of FIG. 1.

FIG. 3A is a schematic view of a recombinant protein, a fusion protein,of the ionic conjugates of FIG. 1.

FIG. 3B is diagrammatic view of an ionizable linking group protrudingfrom the surface of the recombinant protein of FIG. 3A.

FIG. 3C is a schematic view of another recombinant protein of the ionicconjugate of FIG. 1.

FIGS. 4A-4B are a detailed view of the ionic conjugate shown in FIG. 1.

FIG. 5 includes absorption and photoluminescence spectra of solutionscontaining CdSe—ZnS semiconducting particles and CdSe—ZnS semiconductingparticles coated with MBP-zipper.

FIGS. 6A-6C shows cross-sections (˜15 mm thick each) of thin filmsolutions of CdSe—ZnS nanoparticles coated with MBP-leucine zipperrecombinant proteins (a); uncoated CdSe—ZnS nanoparticles (b); CdSe—ZnSnanoparticles coated with IgG using a covalent cross-linking agent EDAC(c).

FIG. 7A is a graph of photoluminescence as a function of the number ofelectrostatically bound proteins.

FIG. 7B is a graph of photoluminescence as a function of pH.

Note that like reference symbols in the various drawings indicate likeelements.

DETAILED DESCRIPTION

The ionic conjugates include an inorganic particle electrostaticallyassociated with a macromolecule. The macromolecule can be selected tointeract with a predetermined species. As a result, the ionic conjugatescan be used in assays to detect the presence of or to quantify theamounts of specific compounds, detect specific interactions ofbiological systems, detect specific biological processes, detectalterations in specific biological processes, or detect alterations inthe structure of specific compounds.

Referring to FIG. 1, an ionic conjugate 10 includes an inorganicparticle 20 and a macromolecule 40 each of which include a linking group30 and 50, respectively, to associatively bind particle 20 tomacromolecule 40 at ends 31 and 51. Ends 31 and 51 are charged orionizable, i.e., the ends can contain localized amounts of positive ornegative charge, and form complementary ionic or electrostatic pairs,such as a partial negative charge on end 31 and a partial positive oncharge 51, or vice versa. In general, macromolecule 40 includes alinking group at any location in or on the macromolecule that isaccessible to interact electrostatically with linking group 30 to attachthe macromolecule to inorganic particle 20. Typically, the charged orionizable portion of linking group 50 extends (protrudes) away from themacromolecule 40.

In general the inorganic particle can be any inorganic materialexhibiting a distinct physical property that can be used to identifythat material. The physical properties can be, but are not limited to,magnetic properties or optical properties. Optical properties include,but are not limited to, emission such as photoluminescence, absorption,scattering and plasmon resonances. For example, the inorganic particlecan be illuminated with a light source at an absorption wavelength tocause an emission at an emission wavelength that can be used todistinguish the emitting material from other materials.

Examples of inorganic particles include, but are not limited to,inorganic colloids and semiconducting nanoparticles. The particles canbe metallic or magnetic particles. The particles also can be crystallineparticles. Examples of inorganic colloids include Ag, Au, or a phosphor.The phosphor can be a inorganic phosphor, such as a rare earth oxide.The inorganic colloids can exhibit distinct reflectivity and scatteringproperties, plasmon resonances, to radiation depending on the size ofthe particles in the colloid. Examples of semiconducting nanoparticlesinclude, but are not limited to, elements from groups II-VI, III-V, andIV of the periodic table. Elements from these groups include, but arenot limited to, CdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, MgTe, GaAs, GaP, GaSb,GaN, HgS, HgSe, HgTe, InAs, InP, InSb, InN, AlAs, AlP, AlSb, AlS, PbS,PbSe, Ge, Si, or an alloy or mixture thereof, including ternary andquaternary mixtures. The semiconducting nanoparticles can besemiconducting nanocrystals. The nanocrystals can be illuminated with alight source at an absorption wavelength to cause an emission at anemission wavelength. The emission has a frequency that corresponds tothe band gap of the quantum confined semiconductor material. The bandgap is a function of the size of the nanocrystal. Nanocrystals havingsmall diameters can have properties intermediate between molecular andbulk forms of matter. For example, nanocrystals based on semiconductormaterials having small diameters can exhibit quantum confinement of boththe electron and hole in all three dimensions, which leads to anincrease in the effective band gap of the material with decreasingcrystallite size. Consequently, both the optical absorption and emissionof nanocrystals shift to the blue (i.e., to higher energies) as the sizeof the crystallites decreases.

The emission from the nanocrystal can be a narrow Gaussian emission bandthat can be tuned through the complete wavelength range of theultraviolet, visible, or infrared regions of the spectrum by varying thesize of the nanocrystal, the composition of the nanocrystal, or both.For example, CdSe can be tuned in the visible region and InAs can betuned in the infrared region. The narrow size distribution of apopulation of nanocrystals can result in emission of light in a narrowspectral range. The population can be monodisperse and can exhibit lessthan a 15% rms deviation in diameter of the nanocrystals, preferablyless than 10%, more preferably less than 5%. Spectral emissions in anarrow range of no greater than about 75 nm, preferably 60 nm, morepreferably 40 nm, and most preferably 30 nm full width at half max(FWHM) can be observed. The breadth of the emission decreases as thepolydispersity of nanocrystal diameters decreases. Semiconductornanocrystals can have high emission quantum efficiencies such as greaterthan 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%.

The semiconductor forming the nanocrystals can include Group II-VIcompounds, Group II-V compounds, Group III-VI compounds, Group III-Vcompounds, Group IV-VI compounds, Group I-III-VI compounds, GroupII-IV-VI compounds, and Group II-IV-V compounds, for example, ZnS, ZnSe,ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, AlN, AlP, AlAs, AlSb, GaN, GaP,GaAs, GaSb, GaSe, InN, InP, InAs, InSb, TIN, TlP, TlAs, TlSb, PbS, PbSe,PbTe, or mixtures thereof.

Methods of preparing monodisperse semiconductor nanocrystals includepyrolysis of organometallic reagents, such as dimethyl cadmium, injectedinto a hot, coordinating solvent. This permits discrete nucleation andresults in the controlled growth of macroscopic quantities ofnanocrystals. Preparation and manipulation of nanocrystals aredescribed, for example, in U.S. application Ser. No. 08/969,302,incorporated herein by reference in its entirety. The method ofmanufacturing a nanocrystal is a colloidal growth process. Colloidalgrowth occurs by rapidly injecting an M donor and an X donor into a hotcoordinating solvent. The injection produces a nucleus that can be grownin a controlled manner to form a nanocrystal. The reaction mixture canbe gently heated to grow and anneal the nanocrystal. Both the averagesize and the size distribution of the nanocrystals in a sample aredependent on the growth temperature. The growth temperature necessary tomaintain steady growth increases with increasing average crystal size.The nanocrystal is a member of a population of nanocrystals. As a resultof the discrete nucleation and controlled growth, the population ofnanocrystals obtained has a narrow, monodisperse distribution ofdiameters. The monodisperse distribution of diameters can also bereferred to as a size. The process of controlled growth and annealing ofthe nanocrystals in the coordinating solvent that follows nucleation canalso result in uniform surface derivatization and regular corestructures. As the size distribution sharpens, the temperature can beraised to maintain steady growth. By adding more M donor or X donor, thegrowth period can be shortened.

The M donor can be an inorganic compound, an organometallic compound, orelemental metal. M is cadmium, zinc, magnesium, mercury, aluminum,gallium, indium or thallium. The X donor is a compound capable ofreacting with the M donor to form a material with the general formulaMX. Typically, the X donor is a chalcogenide donor or a pnictide donor,such as a phosphine chalcogenide, a bis(silyl) chalcogenide, dioxygen,an ammonium salt, or a tris(silyl) pnictide. Suitable X donors includedioxygen, bis(trimethylsilyl) selenide ((TMS)₂Se), trialkyl phosphineselenides such as (tri-n-octylphosphine) selenide (TOPSe) or(tri-n-butylphosphine) selenide (TBPSe), trialkyl phosphine telluridessuch as (tri-n-octylphosphine) telluride (TOPTe) orhexapropylphosphorustriamide telluride (HPPTTe),bis(trimethylsilyl)telluride ((TMS)₂Te), bis(trimethylsilyl)sulfide((TMS)₂S), a trialkyl phosphine sulfide such as (tri-n-octylphosphine)sulfide (TOPS), an ammonium salt such as an ammonium halide (e.g.,NH₄Cl), tris(trimethylsilyl) phosphide ((TMS)₃P), tris(trimethylsilyl)arsenide ((TMS)₃As), or tris(trimethylsilyl) antimonide ((TMS)₃Sb). Incertain embodiments, the M donor and the X donor can be moieties withinthe same molecule.

A coordinating solvent can help control the growth of the nanocrystal.The coordinating solvent is a compound having a donor lone pair that,for example, has a lone electron pair available to coordinate to asurface of the growing nanocrystal. Solvent coordination can stabilizethe growing nanocrystal. Typical coordinating solvents include alkylphosphines, alkyl phosphine oxides, alkyl phosphonic acids, or alkylphosphinic acids, however, other coordinating solvents, such aspyridines, furans, and amines may also be suitable for the nanocrystalproduction. Examples of suitable coordinating solvents include pyridine,tri-n-octyl phosphine (TOP) and tri-n-octyl phosphine oxide (TOPO).Technical grade TOPO can be used.

Size distribution during the growth stage of the reaction can beestimated by monitoring the absorption line widths of the particles.Modification of the reaction temperature in response to changes in theabsorption spectrum or emission spectrum of the particles allows themaintenance of a sharp particle size distribution during growth.Reactants can be added to the nucleation solution during crystal growthto grow larger crystals. By stopping growth at a particular nanocrystalaverage diameter and choosing the proper composition of thesemiconducting material, the emission spectra of the nanocrystals can betuned continuously over the wavelength range of 400 nm to 800 nm. Thenanocrystal has a diameter of less than 150 Å. A population ofnanocrystals has average diameters in the range of 15 Å to 125 Å.

The nanocrystal can be a member of a population of nanocrystals having anarrow size distribution. The nanocrystal can be a sphere, rod, disk, orother shape. The nanocrystal can include a core of a semiconductormaterial. The nanocrystal can include a core having the formula MX,where M is cadmium, zinc, magnesium, mercury, aluminum, gallium, indium,thallium, or mixtures thereof, and X is oxygen, sulfur, selenium,tellurium, nitrogen, phosphorus, arsenic, antimony, or mixtures thereof.

The core can have an overcoating on a surface of the core. Theovercoating can be a semiconductor material having a compositiondifferent from the composition of the core. The overcoat of asemiconductor material on a surface of the nanocrystal can include aGroup II-VI compounds, Group II-V compounds, Group III-VI compounds,Group III-V compounds, Group IV-VI compounds, Group compounds, GroupII-IV-VI compounds, and Group II-IV-V compounds, for example, ZnS, ZnO,ZnSe, ZnTe, CdS, CdO, CdSe, CdTe, MgS, MgSe, HgO, HgS, HgSe, HgTe, AlN,AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, GaSe, InN, InP, InAs, InSb, TIN,TIP, TlAs, TlSb, PbS, PbSe, PbTe, SiO₂, or mixtures thereof. Forexample, ZnS, ZnSe or CdS overcoatings can be grown on CdSe or CdTenanocrystals. An overcoating process is described, for example, in U.S.application Ser. No. 08/969,302, incorporated herein by reference in itsentirety. By adjusting the temperature of the reaction mixture duringovercoating and monitoring the absorption spectrum or absorptionspectrum of the core, over coated materials having high emission quantumefficiencies and narrow size distributions can be obtained.

The particle size distribution can be further refined by size selectiveprecipitation with a poor solvent for the nanocrystals, such asmethanol/butanol as described in U.S. application Ser. No. 08/969,302,incorporated herein by reference. For example, nanocrystals can bedispersed in a solution of 10% butanol in hexane. Methanol can be addeddropwise to this stirring solution until opalescence persists.Separation of supernatant and flocculate by centrifugation produces aprecipitate enriched with the largest crystallites in the sample. Thisprocedure can be repeated until no further sharpening of the opticalabsorption spectrum is noted. Size-selective precipitation can becarried out in a variety of solvent/nonsolvent pairs, includingpyridine/hexane and chloroform/methanol. The size-selected nanocrystalpopulation can have no more than a 15% rms deviation from mean diameter,preferably 10% rms deviation or less, and more preferably 5% rmsdeviation or less.

The outer surface of the nanocrystal can include a layer of compoundsderived from the coordinating solvent used during the growth process.The surface can be modified by repeated exposure to an excess of acompeting coordinating group to form an overlayer. For example, adispersion of the capped nanocrystal can be treated with a coordinatingorganic compound, such as pyridine, to produce crystallites whichdisperse readily in pyridine, methanol, and aromatics but no longerdisperse in aliphatic solvents. Such a surface exchange process can becarried out with any compound capable of coordinating to or bonding withthe outer surface of the nanocrystal, including, for example,phosphines, thiols, amines and phosphates. The nanocrystal can beexposed to short chain polymers which exhibit an affinity for thesurface and which terminate in a moiety having an affinity for asuspension or dispersion medium. Such affinity improves the stability ofthe suspension and discourages flocculation of the nanocrystal.

Transmission electron microscopy (TEM) or small angle x-ray scattering(SAXS) can provide information about the size, shape, and distributionof the nanocrystal population. Powder x-ray diffraction (XRD) patternscan provided the most complete information regarding the type andquality of the crystal structure of the nanocrystals. Estimates of sizeare also possible since particle diameter is inversely related, via theX-ray coherence length, to the peak width. For example, the diameter ofthe nanocrystal can be measured directly by transmission electronmicroscopy or estimated from x-ray diffraction data using, for example,the Scherrer equation. It also can be estimated from the UV/Visabsorption spectrum.

A macromolecule can be any, organic or inorganic, species including acharged, chargeable, or ionizable group. The charged, chargeable, orionizable group can be, but is not limited to, a carboxylate, athiocarboxylate, an amide, a hydrazine, a sulfonate, a sulfoxide, asulfone, a sulfite, a phosphate, a phosphonate, a phosphonium ion, analcohol, a thiol, an amine, an ammonium, a quarternary ammonium, analkyl ammonium, or a nitrate. For example, the ionizable group can be anacidic or basic side chain of an amino acid such as lysine, arginine,histidine, aspartate, or glutamate. The macromolecule can include aplurality of ionizable groups such in polylysine, poly (acrylic acid)(PAA), poly (allyl amine hydrochloride) (PAH), sulfonated polystyrene(SPS), and polydiallyldimethylammonium chloride (PDADMAC). Themacromolecule can be a polypeptide or a polynucleotide.

The macromolecule can exhibit a specific interaction with a separatemolecule or biological target. For example, the macromolecule caninclude a protein, antibody, DNA, RNA, or cell membrane, which binds,interacts, or complexes with a specific compound. Alternatively, amacromolecule that does not exhibit a desired affinity for apredetermined species can be attached to a chemical or biologicalmoiety, such as a protein, antibody, DNA, RNA, or cell membrane, thatexhibits the desired interaction. For example, the macromolecule can beattached to the chemical or biological moiety by biological processes,e.g., from cultures of recombinant organisms (bacteria, yeast, insect,or mammalian cells), or, alternatively, by totally synthetic orsemi-synthetic methods. The biological moiety, whether RNA, cDNA,genomic DNA, vectors, viruses or hybrids thereof, may be isolated from avariety of sources, genetically engineered, amplified, and/or expressedrecombinantly. Any recombinant expression system can be used, including,in addition to bacterial cells, e.g., mammalian, yeast, insect or plantcell expression systems. Examples of biological moieties include maltosebinding protein (MBP) and immunoglobulin G binding protein (Protein G).

Alternatively, nucleic acids can be synthesized in vitro by well-knownchemical synthesis techniques, as described in, e.g., Carruthers (1982)Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am.Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444;Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994)Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown(1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S.Pat. No. 4,458,066. Double stranded DNA fragments may then be obtainedeither by synthesizing the complementary strand and annealing thestrands together under appropriate conditions, or by adding thecomplementary strand using DNA polymerase with an appropriate primersequence.

Techniques for the manipulation of nucleic acids, such as, e.g.,generating mutations in sequences, subcloning, labeling probes,sequencing, hybridization and the like are well described in thescientific and patent literature, see, e.g., Sambrook, ed., MolecularCloning: a Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring HarborLaboratory, (1989); Current Protocols in Molecular Biology, Ausubel, ed.John Wiley & Sons, Inc., New York (1997); Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization With Nucleic AcidProbes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed.Elsevier, N.Y. (1993). Techniques for inserting a charged or ionizablemacromolecule, such as leucine zippers, are also discussed in “Peptide‘Velcro’: Design of a Heterodimeric Coiled Coil,” Current Biology 3(10),658-667 (1993), by O'Shea et al. and in “Fiber-Optic FluorometricSensing of Polymerase Chain Reaction-Amplified DNA Using an ImmobilizedDNA Capture Protein,” Analyt. Biochemistry 235, 61-72 (1996), by Mauroet al.

Recombinant (fusion) proteins can be prepared in a constructed plasmid(double stranded DNA) using appropriate cloning strategies, a techniquefamiliar in bioengineering manipulation of molecules. The cloning andthe protein expression steps can be carried out in Escherichia coli (E.coli), which also serves as the growth environment. Appropriate andspecific enzymes (restriction endonucleases) can be introduced to cutthe plasmid at the proper polylinker cloning site where the gene withthe specific function is introduced. A linking group such as a peptidetail can then be cloned on the carboxy terminal end of the coding regionof the biological moieties having a desired function. Biologicalmoieties include the maltose binding protein (MBP), which binds to thesugar maltose with high affinity, and protein G, which is known tospecifically bind, through its b-subunit, to the Fc region ofimmoglobulin G (IgG). The biological moieties also can be furthermutated such that the moieties exhibits a desired function, such asbinding to a specific biological target. For instance, protein G can bemodified both to interact with a specific molecule and to incorporate aleucine zipper functionality. Protein G then can be electrostaticallyattached to the inorganic particle and used to detect interactions withthe specified biological target.

A synthetic method for attaching the macromolecule to a biologicalmoiety can be performed via known solid-phase peptide couplingtechnology. For example the macromolecule can be formed of a singlecovalently-linked polypeptide chain including an charged or ionizableportion and a portion exhibiting biological specificity. Syntheticmethods for producing the macromolecule can include processes in whichall or part of either or both constituent polypeptides, i.e., thecharged or ionizable and biologically specific portions, forming themacromolecule are prepared using in vitro synthesis. Alternatively, allor part of either or both constituent polypeptide chains can be preparedusing the above recombinant organism(s). For example, the charged orionizable portion of a polypeptide can be produced synthetically and thebiologically specific portion can be obtained by recombinant orsynthetic methods. If the charged/ionizable and biologically specificportions are obtained independently, they can be attached by a) achemical means after chemically activating the peptide termini, such aswith EDC (1-ethyl-3,3-dimethylaminopropyl carbiimide) coupling, b)enzymatically assisted catalysis of activated or unactivatedpolypeptides, c) formation of disulfide (S—S) bonds promoted byoxidation utilizing O₂ or by additional chemical or enzymatic means, ord) by utilizing non-covalent electrostatic or hydrophobic interactionsseparate from the self-assembly interactions used to electrostaticallyattach a macromolecule to a particle. The synthetic methods also includeattaching charged or ionizable portions by any of the above methods topolynucleic acid aptamers (DNA or RNA), peptide nucleic acid (PNA)oligomer, oligosaccharides, lipopolysaccharide, polydextrins, cyclicpolydextrins, crown ethers or similar derivatives, and other natural orsynthetic “receptor” species.

In alternative synthetic approach, a self-assembled complex can beformed via an electrostatic conjugation of a positively chargedpolyelectrolyte, such as polydiallydimethlyammonium chloride, with anegatively charged phosphine-carboxylic acid complex, such as tris2-carboxyethylphosphine. The resulting complex can be coupled to theparticles, with the phosphine groups providing a tether to bind to theparticle surface, while the positively charged polymer facilitatesparticle water-soluble. The charged polymers can include terminal amineor hydroxyl groups, or can be copolymerized with an amino or hydroxylgroup containing monomer, such as allyl amine and hydroxymethacrylate.Standard EDC-type coupling chemistry can be used to effectuate alinkage, such as via a peptide bond, between the amino or hydroxylgroups and a biological moiety to produce a self-assembled ionicconjugate that exhibits an specific biological affinity.

Referring to FIG. 2A, in some embodiments, the inorganic particle is asemiconducting nanocrystal 100 including a semiconducting core 110 and aovercoat 120 encapsulating the core. Semiconducting core 110 andovercoat 120 are made of the semiconducting elements described above. Aplurality of linking groups 130 attach to a surface of overcoat 120 viaa surface interactive group 132 which associates with the materials ofthe overcoat or nanocrystal. Typically, the band gap energy of theovercoat material is larger than the band gap energy of the core. Eachlinking group 130 also contains a charged or ionizable group 136tethered to surface interactive group 132 by a spacer 134. In general,spacer 134 is long enough to prohibit electron charge transfer betweengroups 132 or 120 and 136.

Referring to FIG. 2B, in other embodiments, an inorganic particle 200 ofthis invention is an inorganic colloid particle 210 having a pluralityof linking groups 220 attached to a surface of particle 210 via asurface interactive group 222. Linking group 220 is similar to linkinggroup 130 described above and includes a charged or ionizable group 226and a spacer 224.

In general, the inorganic particles of this invention have a diameterbetween about 10 Å and about 1000 Å; preferably between about 10 Å andabout 500 Å; and most preferably between about 10 Å and about 250 Å. Thesize dispersion about a mean size of the inorganic particles, typically,is less than about 20%; preferably less than about 15%; and morepreferably less than about 10%; and most preferably less than about 5%.In general, more than about 40%; preferably more than about 50% fall;and most preferably more than about 60% of the particles fall within aspecified particle size range.

The surface interactive group can be any chemical moiety having elementsor chemical groups contained therein that are capable of binding to thesurface of the inorganic particle. For example, the surface group caninclude S, N, P, O, or O═P groups. Charged or ionizable groups 136 and226 can include any ionizable chemical group or any chemical grouphaving a native charge, such as a quarternary ammonium group. Examplesof charged or ionizable chemical groups include, but are not limited to,hydroxides, alkoxides, carboxylate, sulfonate, phosphate, phosphonate,quaternary ammonium, and the like.

Linking groups 30, 130, 220 also help to form a water-solubilizing layeraround the particles. In certain embodiments, the surface of theinorganic particle includes a plurality of linking groups, some of whichhelp to water-solubilize the particle and do not electrostaticallyassociate with macromolecules, and others which do. The linking groupsalso can make the particles more stable (i.e., the particles can be usedin dilute concentrations). For example, a monodentate linking group canbe put on the surface of the particle, and then by self-assembly, amonolayer of an oligomer can be wrapped around the particle toeffectively cross-link the functional groups on the surface of theparticle. Self-assembly permits control of assembling the ionicconjugates at the nanometer scale and is similar to the technique oflayer-by-layer self-assembly (or sequential adsorption) used to assemblelarge synthetic and biological polymers. See for example“Molecular-Level processing of Conjugated Polymers 0.1. Layer-by-LayerManipulation of Conjugated Polyions,” Macromolecules 28, 7107 (1995), byM. Ferreira and M. F. Rubner; “New Nanocomposite Films for Biosensors:Layer-by-Layer Adsorbed Films of Polyelectrolytes, Proteins or DNA,”Biosensors & Bioelectronics 9, 677-684 (1994), by Decher et al.; and“Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites,”Science 277: 1232-1237 (1997), by G. Decher.

In preferred embodiments linking groups 30, 130, and 220 have theformula:

(R₁)_(a)—R₂—[(R₃)_(b)(R₄)_(c)]_(d)

wherein (R₁)_(a) is one or more surface interactive groups, R₂ is thespacer, and [(R₃)_(b)(R₄)_(c)]_(d) is the charged or ionizable group;

R₁ is selected from the group consisting of C1-C100 heteroalkyl, C2-C100heteroalkenyl, heteroalkynyl, —OR, —SH, —NHR, —NR′R″, —N(O)HR,—N(O)R′R″, —PHR, —PR′R″, —P(NR′R″)NR′R″, P(O)R′R″, P(O)(NR′R″)NR′R″,—P(O)(OR′)OR″, P(O)OR, P(O)NR′R″, —P(S)(OR′)OR″, and P(S)OR, wherein R,R′, W′ are independently selected from the group consisting of H, abranched or unbranched C1-C100 alkyl, a branched or unbranched C2-C100alkenyl, a branched or unbranched C2-C100 alkynyl, a branched orunbranched C1-C100 heteroalkyl, a branched or unbranched C2-C100heteroalkenyl, a branched or unbranched C2-C100 heteroalkynyl, with theproviso that when a is greater than 1 the R₁ groups can be attached tothe R₂ or R₃ groups at the same or different atoms within those groups,the R₁ groups can be the same or different, or the R₁ groups can form asix, seven, eight, nine, or ten membered cycloalkyl, cycloalkenyl,thereocyclic, aryl, heteroaryl, or a six- to thirty-membered crown etheror heterocrown ether;

R₂ is selected from a bond (i.e., R₂ is absent in which case R₁ attachesto R₃), a branched or unbranched C2-C100 alkylene, a branched orunbranched C2-C100 alkenylene, a branched or unbranched C2-C100heteroalkenylene, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic,aryl, and heteroaryl;

R₃ is selected from a branched or unbranched C2-C100 alkylene, abranched or unbranched C2-C100 alkenylene, a branched or unbranchedC2-C100 heteroalkenylene, cycloalkyl, cycloalkenyl, cycloalkynyl,heterocyclic, aryl, and heteroaryl;

R₄ is selected from the group consisting of hydrogen, a carboxylate, athiocarboxylate, and amid, an amine, a hydrazine, a sulfonate, asulfoxide, a sulfone, a sulfite, a phosphate, a phosphonate, aphosphonium ion, an alcohol, a thiol, an amine, an ammonium, an alkylammonium, a nitrate; and

a is 1 to 4, b is 0 to 3, c is 1 to 3, d is 1 to 3, and when d is 2 or 3the R₃ groups can be the same or different or can be linked together toform a five to ten members cycloalkyl, cycloalkenyl, heterocyclic, aryl,or heteroaryl.

In another preferred embodiment, the linking group 130 has the formula:

HS—C₂H₄—CH(SH)—(C₄H₈)—COOH.

Methods for preparing inorganic particles such as semiconducting CdSenanoparticles with or with a overcoat material are discussed, forexample, in “Semiconductor Nanocrystal Colloids: Manganese Doped CadmiumSelenide, (Core)Shell Composites for Biological Labeling, and HighlyFluorescent Cadmium Telluride,” MIT PhD Thesis, September 1999, by F. V.Mikulec, in “(CdSe)ZnS core-shell nanocrystals: Synthesis andcharacterization of a size series of highly luminescentnanocrystallites,” J. Phys. Chem. B 101, 9463-9475 (1997), by Dabbousiet al.; and in “Semiconductor Nanocrystals as Fluorescent BiologicalLabels,” Science 281, 2013-2016 (1998) by Bruchez, Jr., et al.

Referring to FIGS. 3A-3C, a biological moiety 300, e.g., a recombinantprotein having a specific biological function, includes protein 310 anda linking group 320 protruding from the surface of protein 310. Linkinggroup 320 includes a coupling segment 326, such as a poly Asn linker, abridging group 322, such as sulfur, and a tail 324. In certainembodiments, tail 324 is a polypeptide including approximately 30 aminoacid residues. For example, tail can be any polypeptide which is chargedor ionizable, i.e., contains localized amounts of positive or negativecharge. FIG. 3B shows a positively charged tail 324 commonly referred toas a leucine zipper. Other possible tails include, but are not limitedto, polypeptides including arginine or aspartate groups. Typically,proteins having a leucine zipper tend to form dimers to minimizehydrophobic interactions (FIG. 3C). The resulting dimers are a highprobability state for proteins encoded with leucine. Dimer 380, shown inFIG. 3C, includes two separate proteins 310 and 312, which can be thesame (a homodimer) or different (a heterodimer), linked via two bridginggroups 322, e.g., thiol groups. Tails 324 are attached to the surface ofeach protein through coupling group 326 which can be an atom or amolecule, such as a polypeptide. Each of the bridging groups andcoupling groups can be the same or different.

Referring to FIG. 4A, an ionic conjugate 500 includes an inorganicparticle 510 electrostatically attached to proteins 540 via linkinggroups 520 and 530. In general, the biological moieties canelectrostatically attach to the inorganic particle either as dimers ormonomers. The dimer can be a homodimer or a heterodimer. Additionally,particle 510 can be electrostatically associated with several proteinswhich can be identical or different. A particle including differentproteins can be used to simultaneously detect multiple biologicaltargets of interest. Of course, the exact number of proteins attached tothe particle depends on the diameter of the particle, the overall lengthof the linking groups, and the size of the protein. As seen in FIG. 4B,the number of proteins, N is given by the relationship:

N∝(4π/3)(r ₂ ³ −r ₁ ³)/V(protein)

Where r₂ and r₁ are shown in FIG. 4B, (r₂ ³−r₁ ³) is the volume of spaceavailable for protein packing and V is the average volume of theproteins attached to the particle. Alternatively, N can be derived fromthe expression:

N=0.65((r ₂ ³ −r ₁ ³)/r _(p) ³)

where r_(p) is the radius of the protein. Typically, the ionic conjugateincludes between about 1 to about 25; preferably about 5 to about 15;and most preferably about 5 to about 10 proteins electrostaticallyattached to an inorganic particle having a diameter of about 19 Å.

In other embodiments, the inorganic particle can include an outer shellmade up of either a) an organic shell, b) a thin silica layer, c) acombination of a and b. The organic shell can be monodentate ormultidentate relative to the surface of the inorganic particles. Theorganic shell may also be polymerized around the particle.

The outer shell can be functionalized with groups that can self-assembleto biological moieties that have been either attached to the biologicalmoiety, self-assemble to the moiety itself, or self-assemble to asynthetic molecule which then binds specifically to a biological moiety.The self-assembly process may be an electrostatic interaction with thesurface group. The self-assembly can be through hydrogen bonding orthrough hydrophobic interactions.

Without further elaboration, it is believed that one skilled in the artcan, based on the description herein, utilize the present invention toits fullest extent. The following specific examples, which describesyntheses, screening, and biological testing of various compounds ofthis invention, are therefore, to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. All publications recited herein, including patents, arehereby incorporated by reference in their entirety.

Synthesis of CdSe—ZnS-Lipoic Acid Nanoparticles:

Linking groups used to make semiconducting nanoparticles water solubleare attached to the surface of the semiconducting particle by exchangingtrioctyiphosphine (TOP)/trioctlyphosphineoxide (TOPO) groups on thesurface of the particles with the desired linking group. A dihydrolipoicacid linking group was prepared by reducing commercially availablelipoic acid (also called thioctic acid) purchased from Aldrich as apowder, with sodium borohydride (NaBH₄). See the procedure in A. F.Wagner, J. Organic Chemistry, 1956, 5079-81. The dihydrolipoic acidlinking group increases the stability of the particle such that themodified particles can be used at much more dilute conditions. Theprocedure for exchanging the surface groups of the particles procedureis described below.

A volume of semiconducting nanoparticles (CdSe—ZnS) prepared using thesynthesis route based on growth and annealing of organometalliccompounds at high temperature (See Dabbousi et al.), was taken from thegrowth solution and precipitated by the addition of methanol. Thenanoparticles included a ZnS-overcoating (5-7 monolayers). The isolatedprecipitate was then redispersed in a minimum volume of approximately1:10 solution of butanol:hexane. Again, the semiconducting nanoparticlesare crashed out by the addition of methanol. The procedure ofprecipitating, redispersing, and precipitating was repeated 2-3 times,until most of the TOP/TOPT groups on the surface of the particle wereremoved. A ten to twenty fold excess (by weight) of the desired linkinggroup was added to the moist precipitate. The mixture was placed in anoil bath (around 60-80° C.) and stirred for approx. 3-12 hours. Thegroup exchange procedure was stopped by removing the mixture from theoil bath. The mixture was diluted by adding a small amount of DMF (forapproximately 200 mg of the desired linking group, 100-200 μl of DMF issufficient). Separately, another solution with a slight molar excess(1.5:1) of potassium t-butoxide in a 1:10 volume of DMF:H₂O is preparedand added to the semiconducting particle/DMF solution prepared above toprotonate the lipoic acid groups. A whitish precipitate resulted, whichwas separated from the rest of the solution by centrifugation. Theprecipitate was readily dispersed in water. The nanocrystal dispersionwas purified (from excess potassium t-butoxide and DMF) by concentratingfrom dilute solutions using an ultra-free centrifugal filtration device(from Millipore with cut off at ˜50,000 daltons), and redispersing inwater. Repeating the operation 3 to 4 times provides clean (a purity of˜95% or better) and stable dispersions in water, which have emissioncharacteristics of the nanocrystals and a PL yield of ˜15-20%.

In other synthetic schemes the base, tetramethylammonium hydroxide canbe substituted for potassium t-butoxide in DMF. The former base thoughcan be stored under air, whilst the latter needs to be kept in an inertatmosphere. The dihydrolipoic acid works best if it totally clear. Ifthe reduction lipoic acid to produce dihydrolipoic acid is not complete,a yellow coloration of the solution will be evident. The yellow solutioncan be distilled (distills at 140° C. under vacuum) to yield a clearsolution of dihydrolipoic acid.

Cloning and Preparation of MBP-Basic Zipper Proteins BACKGROUNDREFERENCES

-   1) For MBP dimer expression in bacteria:-   “Engineering the quaternary structure of an exported protein with a    leucine zipper,” Blondel, A. and Bedouelle, H (1991) Protein Eng.    4(4): 457-61.-   2) For heterodimer formation via expressed recombinant proteins:-   “A general method of facilitating heterodimeric pairing between    proteins: application to expression of alpha and beta T-cell    receptor extracellular segments,” Chang, H. C., Bao, Z., Yao, Y.,    Tse, A. G., Goyarts, E. C., Madsen, M., Kawasaki, E., Brauer, P. P.,    Sachettini, J. C., Nathenson, S. G. et al. (1994) Proc. Natl. Acad.    Sci. USA 91(24): 11408-11412.-   3) For design and basic characterization of the leucine zippers used    in this work:-   “Peptide Velcro: design of a heterodimeric coiled coil,” O'Shea, E.    K., Lumb, K., and Kim, P. S. (1993) Current Biology 3(10): 658-667.-   4) For a description of bioconjugates:-   “Bioconjugation of Highly Luminescent Colloidal CdSe—ZnS Quantum    Dots with an Engineered Two-Domain Recombinant Protein,” H.    Mattoussi et al. (2001) Phys. Stat. Sol. (b), 224(1): 277-283.-   5) For a description of bioconjugates:-   “Self-Assembly of CdSe—ZnS Quantum Dot Bioconjugates Using an    Engineered Recombinant Protein,” H. Mattoussi et al. (2001) J. Am.    Chem. Soc., 122: 12142-12150.

Preparation of the Basic (Positively Charged) Leucine Zipper Gene

Two DNA oligonucleotide primers were synthesized to anneal to the 5′ and3′ ends of the basic leucine zipper in the PCRIIBasic plasmid suppliedby Chang et al.

(SEQ ID NO: 1) Primer 1: 5′-TGCGGTGGCTCAGCTCAGTTG-3′ (SEQ ID NO: 2)Primer 2: 5′-GCTCTAGATTAATCCCCACCCTGGGCGAGTTTC-3′

Using PCR, the basic zipper was amplified using primers 1 and 2 and pfupolymerase to produce a DNA fragment of approximately 120 bp coding forthe basic zipper and with termini suitable for processing prior toinsertion 3′ of the MalE gene and coding for a stop codon and a uniquecysteine residue 5′ of the zipper sequence for eventual covalent dimerformation in the expressed fusion protein. Processing of the amplifiedzipper-encoding fragment was achieved by digesting a portion of the DNAwith restriction endonuclease XbaI to provide an appropriatelyoverlapping 3′ terminus for the subsequent cloning step. The 5′ end ofthe PCR fragment was designed to be blunt ended, so no additionalprocessing was required prior to the next step.

Cloning of the Leucine Zipper Gene onto the C-Terminal Coding Sequencefor MBP

The prepared DNA fragment was then ligated enzymatically into thecommercial vector pMal-c2 (New England Biolabs) that had been processedwith the restriction endonucleases XmnI and XbaI; these enzyme cleavagesites in the DNA vector provided the 5′ blunt and 3′ overlappingsequences required for successful ligation of the prepared basic leucinezipper containing fragment prepared as described. After transformationof E. coli DH5″ with ligation product and obtaining ampicillin resistantbacterial colonies, several colonies were tested for presence of thedesired inserted basic leucine zipper DNA by colony PCR using DNAoligonucleotide primers flanking the vector cloning sites. Severalpositive colonies were chosen for amplification by overnight growth on asmall scale, followed by preparation of small amounts of pMal-BasicZipper DNA.

Expression of the MBP-Basic Zipper Protein in Bacteria

Several candidate plasmids from the above cloning procedure were testedfor the ability to express the desired fusion protein in the DH5″ hostbacterial strain as evaluated by small-scale cell culture (10 ml) andSDS gel electrophoresis of IPTG-induced expression product. Out ofseveral successful clones, one was selected to carry out DNA sequencingto verify the accuracy of the DNA sequence, for further expressionstudies, and ultimately for larger scale expression work. The correctexpected DNA sequence for the selected clone was verified by the MGIFsequencing facility at the University of Georgia.

Additional small-scale expression studies in various bacterial strainswere subsequently conducted to optimize production of the fusionprotein. E. coli BL21, a lon protease deficient strain, proved to besuitable for expression of the protein.

Appending a (His)6 (Hexahistidine) Peptide onto the C-Terminus of theFusion Protein

In order to provide additional flexibility in preparation andpurification of this and other similar fusion proteins, DNA coding for ahexahistidine peptide sequence was appended onto the 3′-end of thepMal-Basic Zipper sequence. Preparing this construct required synthesisof a new 3′ primer used together with primer 1 (above) to allowamplification of a basic zipper DNA fragment lacking the codon fortranslation termination that was implanted in the initial constructdescribed above:

(SEQ ID NO: 3) Primer 3: 5′-GCTCTAGATGAATCCCCACCCTGGGCGAGTTTC-3′.

Following exactly the procedure described above, an intermediateconstruct was made that was identical to the pMal-Basic Zipper exceptfor the lack of a stop codon 3′ of the leucine zipper. DNA coding thisintermediate construct was cleaved with restriction endonucleases XbaIand PstI, and the following synthetically prepared duplex DNA wasenzymatically ligated into these sites:

(SEQ ID NO: 4) 5′-CTAGCGGTCACCACCACCACCACCACTGACTGCA-3′ (SEQ ID NO: 5)3′-GCCAGTGGTGGTGGTGGTGGTGACTG-5′

After transformation of E. coli DH5″ with ligation products of thevector and the indicated insert DNA, colony PCR analysis once more usedto find clones coding for MBP followed in tandem by the basic leucinezipper sequence, the hexahistidine sequence and a translational stopcodon. Expression in E. coli BL21 was again found to yield satisfactoryamounts and quality of protein for larger scale work.

Cell Culture of Either MBP-Basic Zipper or MBP-Basic Zipper-(His)6Protein

A single colony of freshly transformed E. coli BL21 was transferred into10 ml Luria Broth (LB) containing 100 μg/ml carbenicillin and theculture was shaken at 37° C. overnight (approximately 15 hr). 2.0 ml ofthis overnight culture was transferred into 1 liter of LB containing 50μg/ml carbenicillin and 2 grams glucose. After growing to an opticaldensity of 0.6 at 37° C., the flask containing the cell culture wastransferred to shaking at 30° C. for 15 min prior to adding IPTG(isopropylthiogalactopyranoside) to a final concentration of 1 mM. After2 hr shaking at this temperature, the cells were sedimented at 4° C. bycentrifugation. The resulting cell pellet was quick frozen in powdereddry ice and stored at −80° C. until thawing for purification.

Purification of MBP-Basic Zipper-(His)6 from 1 Liter of Cell Culture

Lysis buffer (35 ml of 50 mM HEPES, 0.3 M NaCl, 5 mM imidazole, pH 7.9containing one tablet of Boehringer EDTA-free protease inhibitorcocktail) was added to the tube containing the thawing pellet freshlyremoved from −80° C. storage. After complete resuspension on ice, thecells were lysed by sonication for 5×1 minute in ice water. The lysedcells were centrifuged at 16,000 RPM at 4° C. for 30 min. After thecrude supernatant was passed through a 0.8/0.2 micron dual stage syringeultrafilter, 15 ml of a 50% suspension of NiNta metal chelating resin(Qiagen) equilibrated with lysis buffer was added, and the tube tumbledat 4° C. for 1 hr. The resin and bound protein was briefly centrifugedand the supernatant discarded; the resin was then washed 2× with 40 mllysis buffer. The washed resin was poured into a 1.5 cm diameter glasschromatography at 4° C., and 50 ml lysis buffer (sans proteaseinhibitors) was passed over the column at 0.8 ml/min, followed by 90 mlwash buffer (50 mM HEPES, 0.3 M NaCl, 20 mM imidazole, pH 7.9). Theproduct is eluted from the washed column with elute buffer (50 mM HEPES,0.3 M NaCl, 250 mM imidazole, pH 7.4). Pooled fractions containingprotein were then applied to a 25 ml packed bed (2 cm diameter) columnof immobilized amylose (4° C.) previously equlibrated with 50 mM HEPES,0.1 M NaCl, pH 7.4 at ca. 1 ml/min. The column was washed with 100 ml ofthe above buffer, then the protein was eluted with this buffercontaining 10 mM maltose. Collected fractions containing purifiedprotein were pooled and passed through a sterile 0.45 micron syringefilter and stored at 4° C. Purified protein was analyzed by SDS gelelectrophoresis+/−dithiothreitol reducing agent (15 mg/ml in boiledsamples) to evaluate the degree of dimer formation.

Maltose Binding Protein Ionic Conjugate

The MBP-leucine zipper fusion protein was electrostatically attached tothe CdSe(core)-ZnS(overcoat)-dihydrolipoic acid modified semiconductingparticles by mixing the fusion protein and inorganic particles in boratebuffered solutions at pH ˜8-9. A pH greater than about 7 is mostsuitable for protein manipulation by preserving the nanocrystalsolubility and imparting negative surface charge coverage. Simpleaddition of the MBP-leucine zipper fusion protein to a solutioncontaining a fixed amount of nanocrystals in buffered solution yieldedionic bio-conjugates free of aggregates, irrespective of the mole ratioof inorganic particles to proteins. Typically, the ratio of inorganicparticles to proteins is between 1 and 10. The inorganic particles hadan average diameter of about 19 Å.

The advantage of surface modification using the present method is itssimplicity and versatility. It is only necessary to add the fusionprotein to the particles to coat, and the desired protein attaches tothe surface nearly instantaneously. Furthermore, in some instancescoating of the inorganic particles with the biological moieties resultsin enhancement of the photoluminescence yield. FIG. 5 shows thatparticles coated with the MBP-zipper protein have approximately athree-fold increase in luminescence intensity relative to particleswhich are not electrostatically associated with a biological moiety.

The MBP-zipper coated nanocrystals (and particles) prepared vianoncovalent cross-linking were also examined using fluorescence confocalmicroscopy imaging. FIGS. 6A-6C show cross-sectional images using alaser scanning confocal microscope of solutions of MBP-Zipper bound tonanocrystals (core-overcoat with 19 Å core radius), along with an imageof a sample of the same particles covalently cross-linked to ovalbuminvia such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDAC or EDC). Covalent cross-linking procedure are discussed in“Semiconductor Nanocrystals as Fluorescent Biological Labels,” Science281, 2013-2016 (1998), by Bruchez, Jr. et al.; “Quantum DotBioconjugates for Ultrasensitive Nonisotopic Detection,” Science 281,2016-2018, by W. C. W. Chan, and S, Nie; and references cited therein.Significant aggregates (that manifest in a the appearance ofconstellation-like features) were formed when using the covalent method(FIG. 6C), whereas ionic conjugates of inorganic particles coated withthe MBP-zipper appeared identical to uncoated material (See FIGS. 6A and6B). The emission recorded in FIGS. 6A and 6B results from singleparticles and is characteristic of a stable and aggregation-freesolutions. Additionally, the photoluminescence of the inorganicparticles coated with the MBP-zipper (FIG. 6A) was increased relative touncoated inorganic particles (FIG. 6B) for thin films containing thecomparable concentration of particles. For each image, the solution ofionic bioconjugates was excited at 488 nm, i.e., below the location ofthe first absorption peak of the nanoparticles. Appropriate filters wereused to block out the excitation signal. Images of nanoparticles coatedwith ovalbumin using the covalent (EDAC) binding approach were alsorecorded along with images of pure nanoparticle (protein-free) andnanoparticles complexed to fusion proteins. Each image represents verythin slices (˜15 micron thick and an area of 150×100 micron² each) alongthe laser path (exciting signal to traveling vertically). The lastimages in the bottom of the figure represent the cut adjacent to thesupport surface (bottom of the optical dish). The green bright spots onthe images represent luminescence emission from individual nanoparticlesdispersed in the solution film. The results described above indicatethat the present coating approach is effective in providingaggregation-free (even at very small scales) nanocrystal-proteinbioconjugates. The samples are stable over a long period of time(months). The aggregates (FIG. 6C) precipitate to the bottom of thesample by gravity, and leave non-reacted particles floating in thesolution, which may give the false impression that covalently coatedparticles are stable and do not encounter large scale aggregation.Aggregation was even more pronounced in solutions where IgG is attachedto the nanocrystals via EDAC (data not shown).

Referring to FIGS. 7A and 7B, the effects of pH and the number ofprotein on the nanoparticle were tested. At pH of about 9, thephotoluminescence increases as the number of fusion proteinselectrostatically attached to the nanoparticle is increased. At aconstant number of proteins complexed to the nanoparticles, thephotoluminescence increased with increasing pH.

In a subsequent experiment, the bioactivity of the ionic conjugatesincluding the MBP was tested by passing the ionic conjugates through acolumn of amylose functionalized resins. The ionic conjugates bound tothe resin as MBP interacted with amylose. Thus, the ionic conjugatesmaintained their bioactivity. Ionic conjugates bound to the amylosefunctionalized resins were released by washing the column with a maltosesolution.

Cloning and Preparation of G-Basic Zipper Proteins

The coding sequence for the IgG binding b2 sub-domain of streptococcalprotein G (PG) was cloned and expressed in the E. coli cloning vectorpBad/HisB (an inducible expression vector from Invitrogen). The linkerplus the leucine zipper tail were inserted downstream at the site 3′away from the PG. In addition, a polyhistidine short chain(hexahistidine) was attached to the end of the leucine tail tofacilitate purification of the final product. These successive genemanipulations provided a fusion streptococcal protein G that has an IgGb2 binding sub-domain and a leucine zipper charged tail, which plays amajor role in the present coating scheme.

Ionic conjugates including semiconducting nanoparticles (CdSe—ZnS)coated with protein G-zipper were synthesized via the method describedabove. However, the ratio of inorganic particles to biomolecules,typically, is less than the ratio of MBP-proteins to inorganicparticles.

The resulting ionic bioconjugates were passed through a columncontaining resins functionalized with IgG and allowed to react forapproximately 15 minutes. Washing the column with pure buffer solutionresulted in the release of negligible amounts of ionic bio-conjugates.The functionalized column retained approximately 95-98% of the ionicbioconjugates. The percentage of ionic conjugates contained within thecolumn was determined by monitoring the effluent from the column forluminescence indicative of the semiconducting particles. These resultsimply that protein G-zipper molecules bind to the inorganic particles.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1-46. (canceled)
 47. A composition comprising: a semiconductornanocrystal with a linking group having a distal end and a proximal end,wherein the distal end is bound to an outer surface of the semiconductornanocrystal, and the proximal end comprises a first charged or ionizablemoiety; and a protein or polypeptide including a first plurality ofcharged or ionizable moieties, wherein the first charged or ionizablemoiety and the first plurality of charged or ionizable moietiesassociate the semiconductor nanocrystal with the protein or polypeptide,and wherein the protein or polypeptide comprises a plurality ofhistidine residues.
 48. The composition of claim 47, wherein the firstplurality of charged or ionizable moieties includes the plurality ofhistidine residues.
 49. The composition of claim 48, wherein theplurality of histidine residues includes a plurality of sequentialhistidine residues.
 50. The composition of claim 49, wherein theproximal end comprises a first charged moiety.
 51. The composition ofclaim 49, wherein the proximal end comprises a hydroxide, an alkoxide, acarboxylate, a sulfonate, a phosphate, or a phosphonate.
 52. Thecomposition of claim 51, wherein the proximal end comprises acarboxylate.
 53. The composition of claim 49, wherein the plurality ofhistidine residues is appended to a terminus of the protein orpolypeptide.
 54. The composition of claim 49, wherein the plurality ofhistidine residues includes a polyhistidine short chain.
 55. Thecomposition of claim 54, wherein the polyhistidine short chain includeshexahistidine.
 56. A method of making composition, comprising: providinga semiconductor nanocrystal with a linking group having a distal end anda proximal end, wherein the distal end is bound to an outer surface ofthe semiconductor nanocrystal, and the proximal end comprises a firstcharged or ionizable moiety; and contacting a protein or polypeptideincluding a first plurality of charged or ionizable moieties with thesemiconductor nanocrystal, wherein the first charged or ionizable moietyand the first plurality of charged or ionizable moieties associate thesemiconductor nanocrystal with the protein or polypeptide, and whereinthe protein or polypeptide comprises a plurality of histidine residues.57. The method of claim 56, wherein the first plurality of charged orionizable moieties includes the plurality of histidine residues.
 58. Themethod of claim 57, wherein the plurality of histidine residues includesa plurality of sequential histidine residues.
 59. The method of claim58, wherein the proximal end comprises a first charged moiety.
 60. Themethod of claim 58, wherein the proximal end comprises a hydroxide, analkoxide, a carboxylate, a sulfonate, a phosphate, or a phosphonate. 61.The method of claim 60, wherein the proximal end comprises acarboxylate.
 62. The method of claim 58, wherein the plurality ofhistidine residues is appended to a terminus of the protein orpolypeptide.
 63. The method of claim 58, wherein the plurality ofhistidine residues includes a polyhistidine short chain.
 64. The methodof claim 63, wherein the polyhistidine short chain includeshexahistidine.